Msed_0399 (A4YDS4) notes

Metallosphaera sedula DSM 5348, Msed_0399, UniProt A4YDS4 (TrEMBL, unreviewed;
"3-hydroxyacyl-CoA dehydrogenase, NAD-binding protein"), 651 aa.

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Domain architecture

• UniProt features: 3-hydroxyacyl-CoA dehydrogenase NAD-binding domain (Pfam
  PF02737, residues 2-181), two 3HCDH C-terminal domains (PF00725, 183-277 and
  299-389), and an enoyl-CoA hydratase/isomerase domain (PF00378, 407-650). So the
  dehydrogenase is N-terminal and the crotonase is C-terminal.
• This matches Ramos-Vera et al. PMID:21169482.
• The UniProt SIMILARITY line from ARBA00008750 says the reverse ("In the
  N-terminal section; belongs to the enoyl-CoA hydratase/isomerase family"). This
  contradicts the entry's own Pfam domain coordinates. It is a text-level ARBA
  error that does not reach GO.
• PANTHER PTHR23309:SF49 ("peroxisomal bifunctional enzyme"), the same
  multifunctional-enzyme family as E. coli FadB and human EHHADH. The domain order is
  reversed relative to FadB PMID:21169482.

Biochemistry

• Identification: the enzyme was purified from autotrophically grown cells PMID:21169482. The
  recombinant enzyme catalysed both steps PMID:21169482.
• Cofactor: NAD+, not NADP+ PMID:21169482. The protein
  also binds NAD+ in the purification PMID:21169482.
• So GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity (EC 1.1.1.35)
  fits. GO:0008691 3-hydroxybutyryl-CoA dehydrogenase activity is defined with NADP+
  (EC 1.1.1.157) and does not fit, even though the paper and BRENDA use EC 1.1.1.157.
• The NAD+ binding IEA (InterPro IPR006176) is therefore correct here. This contrasts
  with the default NAD binding error on the NADPH enzyme Msed_0709 (ARCHAEA pattern 6).
• Expression: PMID:21169482. The activity is not autotrophy-specific.
• In-vitro reconstitution: Msed_0406, Msed_1321, Msed_0399 and Msed_0656 PMID:24532060, with NAD+ in the assay.
• Substrate range [PMID:32218776 "In hydratase reaction, Msed_0399 was promiscuous and
  could hydrate not only crotonyl-CoA, but also (E)-2-octenoyl-CoA and acrylyl-CoA
  (Table 2)."; "Similarly, it acted as dehydrogenase with both C4 and C8
  (S)-3-hydroxyacyl-CoAs (Table 2)."; "Interestingly, the enzyme was most active with
  octenoyl-CoA, thus suggesting that Msed_0399 may participate in β-oxidation, in
  addition to autotrophic CO2 fixation."].
• The hydratase therefore acts on (2E)-enoyl-CoAs from C3 to C8. GO:0018812
  3-hydroxyacyl-CoA dehydratase activity ((2E)-enoyl-CoA; no stereochemistry stated)
  is the right level. GO:0120092 (C4 only) is too narrow, and GO:0080023 is the
  (3R)-specific reaction, while this enzyme is (S)-specific.

Physiological role: not the main cycle enzyme

• Dehydrogenase: PMID:32218776. Msed_1423
  (UniProt A4YGM9) clusters with Msed_1424 and Msed_1426. Msed_1423 and Msed_0389 have
  kcat/Km values 3- and 20-fold higher than Msed_0399 for (S)-3-hydroxybutyryl-CoA.
• Conclusion of the same paper: PMID:32218776.
• Distribution: PMID:32218776.
• Genomic context: PMID:32218776. Caveat:
  PMID:32218776.
• Hydratase: PMID:33472982; PMID:33472982. The protein is abundant in autotrophic
  cells (89% of the level of Msed_2001), so its low in-vivo share reflects low Vmax, not
  absence.
• In the dicarboxylate/4-hydroxybutyrate cycle of Ignicoccus and Pyrobaculum, the
  orthologous fusion protein is the only candidate and is thought to do both steps
  (PMID:21169482, PMID:32218776). Its secondary role in M. sedula is a Sulfolobales
  peculiarity.

Curation conclusions

• Core MF: GO:0003857 (NAD+-specific, (S)-specific, C4 to C8 tested) and GO:0018812
  (C-terminal crotonase domain). Both are intrinsic activities of the protein. Neither
  is the main in-vivo catalyst of its 3HP/4HB cycle step.
• Process: no process term is well supported. Carbon fixation (GO:0015977) is not
  added as NEW because the authors who measured the fluxes conclude the enzyme carries
  "a small flux ..., at best", and the gene is missing from several autotrophic
  Sulfolobales. Fatty acid metabolism is a hypothesis from substrate range and gene
  context, with no in-vivo evidence. GO:0006631 is kept as non-core (broad, compatible
  with the C4 to C8 acyl-CoA chemistry). GO:0006635 is defined as complete oxidation of a
  long-chain fatty acid, which has never been tested, so it is UNDECIDED.
• Module impact: the module's step 6 ((S)-3-hydroxybutyryl-CoA oxidation) uses Msed_0399
  as the representative. PMID:32218776 shows that Msed_1423 (A4YGM9) is the main enzyme.
  The step should be re-grounded on Msed_1423, with Msed_0399 at most a minor
  contributor. GO:0003857 remains the right function term, because Msed_1423 is also
  NAD-dependent PMID:32218776.
  PMID:32218776 should be added to the module's evidence.